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Rapid, easy-to-use, and low-cost systems for biological sample testing are important for point-of-care diagnostics and various other health applications. The recent pandemic of Coronavirus Disease 2019 (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) showed an urgent need to rapidly and accurately identify the genetic material of SARS-CoV-2, an enveloped ribonucleic acid (RNA) virus, in upper respiratory specimens from people. In general, sensitive testing methods require genetic material extraction from the specimen. Unfortunately, current commercially available extraction kits are expensive and involve time-consuming and laborious extraction procedures. To overcome the difficulties associated with common extraction methods, we propose a simple enzymatic assay for the nucleic acid extraction step using heat mediation to improve the polymerase chain reaction (PCR) reaction sensitivity. Our protocol was tested on Human Coronavirus 229E (HCoV-229E) as an example, which comes from the large coronaviridae family of viruses that affect birds, amphibians, and mammals, of which SARS-CoV-2 is a member. The proposed assay was performed using a low-cost, custom-made, real-time PCR system that incorporates thermal cycling and fluorescence detection. It had fully customizable reaction settings to allow versatile biological sample testing for various applications, including point-of-care medical diagnosis, food and water quality testing, and emergency health situations. Our results show that heat-mediated RNA extraction is a viable extraction method when compared to commercial extraction kits. Further, our study showed that extraction has a direct impact on purified laboratory samples of HCoV-229E, but no direct impact on infected human cells. This is clinically relevant, as it allows us to circumvent the extraction step on clinical samples when using PCR.

Global health crises due to the prevailing Coronavirus Disease 2019 (COVID-19) pandemic have placed significant strain on health care facilities such as hospitals and clinics around the world. Further, foodborne and waterborne diseases are not only spreading faster, but also appear to be emerging more rapidly than ever before and are able to circumvent conventional control measures. The Polymerase Chain Reaction (PCR) system is a well-known diagnostic tool for many applications in medical diagnostics, environmental monitoring, and food and water quality assessment. Here, we describe the design, development, and testing of a portable, low-cost, and real-time PCR system that can be used in emergency health crises and resource-poor situations. The described PCR system incorporates real-time reaction monitoring using fluorescence as an alternative to gel electrophoresis for reaction analysis, further decreasing the need of multiple reagents, reducing sample testing cost, and reducing sample analysis time. The bill of materials cost of the described system is approximately $340. The described PCR system utilizes a novel progressive selective proportional–integral–derivative controller that helps in reducing sample analysis time. In addition, the system employs a novel primer-based approach to quantify the initial target amplicon concentration, making it well-suited for food and water quality assessment. The developed PCR system performed DNA amplification at a level and speed comparable to larger and more expensive commercial table-top systems. The fluorescence detection sensitivity was also tested to be at the same level as commercially available multi-mode optical readers, thus making the PCR system an attractive solution for medical point-of-care and food and water quality assessment.

Listeria monocytogenes is a foodborne pathogen capable of secreting listeriolysin O (LLO), a pore-forming toxin encoded by the hly gene. While the functions of LLO have been studied extensively, how the production of LLO is modulated by the intestinal environment, devoid of oxygen and enriched in short chain fatty acids (SCFAs), is not completely understood. Using L. monocytogenes strain 10403s, we found that hly transcription was moderately decreased by aerobic SCFA exposures but significantly increased by anaerobic SCFA exposures. Moreover, aerobic, but not anaerobic, exposure to low levels of SCFAs resulted in a significantly higher LLO activity. These results demonstrated that transcriptional and post-transcriptional regulations of LLO production were separately modulated by SCFAs and were responsive to oxygen levels. Examining isogenic mutants revealed that PrfA and SigB play a role in regulating LLO production in response to SCFAs. Effects of SCFAs were also present in the cardiotropic strain 07PF0776 but distinctly different from those in strain 10403s. For both strains, prior exposures to SCFAs altered intracellular infections in Caco-2 and RAW264.7 cells and the plaque sizes in L fibroblasts, a result confirming the ability of L. monocytogenes to adapt to SCFAs in ways that impact its subsequent infection outcomes.

Listeria monocytogenes (L. monocytogenes) is a Gram-positive, enteric pathogen and the causative agent of listeriosis. During transition through the gastrointestinal tract, L. monocytogenes routinely encounters suboxic conditions. However, how the exposure to the low oxygen environment affects subsequent pathogenesis is not completely understood. Our lab previously reported that anaerobically grown L. monocytogenes exhibited an intracellular growth defect in macrophages even though the infection took place under aerobic conditions. This phenotype suggests that prior growth conditions have a prolonged effect on the outcome of subsequent intracellular infection. In this study, to further investigate the mechanisms that contribute to the compromised intracellular growth after anaerobic exposure, we hypothesized that the lack of respiratory activity under anaerobic conditions prevented anaerobically grown L. monocytogenes to establish subsequent intracellular growth under aerobic conditions. To test this hypothesis, respiratory activity in anaerobically grown L. monocytogenes was stimulated by exogenous fumarate and subsequent intracellular pathogenesis was assessed. The results showed that fumarate supplementation significantly increased the respiratory activity of anaerobically grown L. monocytogenes and rescued the subsequent intracellular growth defect, likely through promoting the production of listeriolysin O, phagosomal escape, and cell-cell spread. This study highlights the importance of respiratory activity in L. monocytogenes in modulating the outcome of subsequent intracellular infections.

Propionate is a common food preservative and one of the major fermentation acids in the intestines. Therefore, exposure to propionate is frequent for foodborne pathogens and likely takes place under suboxic conditions. However, it is not clear whether the absence of oxygen affects how pathogens respond to propionate. Here, we investigated how propionate exposure affects Listeria monocytogenes growth and virulence factor production under aerobic or anaerobic conditions and showed that oxygen indeed plays a key role in modulating L. monocytogenes response to propionate. Under aerobic conditions, propionate supplementations had no effect on planktonic growth but resulted in decreased adherent growth. Under anaerobic conditions, propionate supplementations resulted in a pH-dependent inhibition of planktonic growth and increased adherent growth. Cultures grown with propionate accumulated higher levels of acetoin under aerobic conditions but lower levels of ethanol under both aerobic and anaerobic conditions. Metabolic perturbations by propionate were also evident by the increase in straight chain fatty acids. Finally, propionate supplementations resulted in increased listeriolyin O (LLO) production under anaerobic conditions but decreased LLO production under aerobic conditions. These results demonstrate for the first time that the presence or absence of oxygen plays a critical role in shaping L. monocytogenes responses to propionate.